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Lipoprotein Lipase but Not Hormone-Sensitive Lipase Activities Achieve Normality After Surgically Induced Weight Loss in Morbidly Obese Patients

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Background Although bariatric surgery is currently the most common practice for inducing weight loss in morbidly obese patients (BMI > 40 kg/m2), its effect on the lipid content of adipose tissue and its lipases (lipoprotein lipase [LPL] and
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  CLINICAL RESEARCH Lipoprotein Lipase but Not Hormone-Sensitive LipaseActivities Achieve Normality After SurgicallyInduced Weight Loss in Morbidly Obese Patients E. Pardina  &  A. Lecube  &  R. Llamas  &  R. Catalán  & R. Galard  &  J. M. Fort  &  H. Allende  &  V. Vargas  & J. A. Baena-Fustegueras  &  J. Peinado-Onsurbe Received: 13 January 2009 /Accepted: 27 April 2009 /Published online: 20 May 2009 # Springer Science + Business Media, LLC 2009 Abstract  Background   Although bariatric surgery is currently the most common practice for inducing weight loss in morbidly obese patients (BMI>40 kg/m 2 ), its effect on the lipid content of adipose tissue and its lipases (lipoprotein lipase [LPL] andhormone-sensitive lipase [HSL]) are controversial.  Methods  We analyzed LPL and HSL activities and lipidcontent from plasma as well as subcutaneous (SAT) andvisceral (VAT) adipose tissue of 34 morbidly obese patients(MO) before and after (6 and 12 months) Roux-en-Y gastric bypass surgery and compare the values with those of normal weight (control) patients.  Results  LPL activity was significantly higher in MO (SAT=32.9±1.0 vs VAT=36.4±3.3 mU/g tissue;  p <0.001) than incontrol subjects (SAT=8.2±1.4 vs VAT=6.8±1.0 mU/gtissue) in both adipose depots. HSL activity had similar values in both types of tissue (SAT=32.8±1.6 and VAT=32.9±1.6 mU/g) of MO. In the control group, we foundsimilar results but with lower values (SAT=11.9±1.4 vsVAT=12.1±1.4 mU/g tissue). Twelve months after surgery,SAT LPL activity diminished (9.8±1.4 mU/g tissue,  p <0.001 vs morbidly obese), while HSL (46.6±3.7 mU/gtissue) remained high. All lipids in tissue and plasmadiminished after bariatric surgery except plasma nonester- OBES SURG (2009) 19:1150  –  1158DOI 10.1007/s11695-009-9853-3E. Pardina and A. Lecube contributed equally to this study.J.A. Baena-Fustegueras and J. Peinado-Onsurbe share senior authorship.E. Pardina :  R. Llamas :  J. Peinado-Onsurbe ( * )Biochemistry and Molecular Biology Department,Biology Faculty, Universitat de Barcelona,Diagonal 645,08028 Barcelona, Spaine-mail: jpeinado@ub.eduA. LecubeDiabetes Research Unit, Institut De Recerca Vall D ’ Hebron,CIBER de Diabetes y Enfermedades Metabólicas Asociadas(CIBERDEM), Instituto de Salud Carlos III (ISCIII),Barcelona, SpainR. Catalán : R. GalardBiochemistry Department, Institut De Recerca Vall D ’ Hebron,Barcelona, SpainJ. M. Fort  :  J. A. Baena-FusteguerasEndocrinology Surgery Unit, Institut De Recerca Vall D ’ Hebron,Universitat Autònoma De Barcelona,Barcelona, SpainH. AllendePathology Division, Institut De Recerca Vall D ’ Hebron,Universitat Autònoma De Barcelona,Barcelona, SpainV. VargasLiver Unit, Institut De Recerca Vall D ’ HebronUniversitat Autònoma De Barcelona,Barcelona, SpainH. Allende : V. VargasCIBER de Enfermedades Hepáticas y Digestivas (CIBEREHD),Instituto de Salud Carlos III (ISCIII),Barcelona, Spain  ified fatty acids, which maintained higher levels thancontrols (16±3 vs 9±0 mg/dL;  p <0.001, respectively). Conclusions  When obese patients lose weight, they losenot only part of the lipid content of the cells but also thecapacity to store triacylglycerides in SAT depots. Keywords  Adipocytesize.Obesity.Weightloss. NEFA.LPL.HSL Abbreviations HOMA-IR Homeostasis model assessment of insulinresistance NEFA Nonesterified fatty acidLPL Lipoprotein lipaseHSL Hormone-sensitive lipase. Introduction Adipose tissue mass in mammals is maintained by adynamic equilibrium between lipid storage, which ismediated by lipoprotein lipase (LPL), and fat mobilization(lipolysis), which is regulated by hormone-sensitive lipase(HSL). Both enzymes play important roles in obesity [1].However, there is still uncertainty about the importance of each enzyme in the acquisition and maintenance of fat depots, particularly in subcutaneous (SAT) and visceral(VAT) adipose tissue in morbidly obese patients.LPL activity is related to the liberation of lipolytic products from chylomicra and very-low-density lipopro-teins, which are captured by adipocytes and stored astriacylglycerides (TAG). Several authors have found mark-edly elevated LPL activities in adipose tissue of obese patients, but others have reported contrary results. There arealso conflicting data about LPL activity after weight loss [2].Moreover, there are differences in the TAG uptake capacity between the different adipose depots. According to Marin et al., the uptake of labeled TAG is approximately 50% higher in omental adipose tissue than in subcutaneous abdominaladipose tissue in men with a wide variation of body fat.However, these results did not correlate with LPL activity[3]. The increase in visceral fat mass with increasing total body fat could be explained by an increase in fat cell sizeonly up to a certain adipocyte weight. Further elevations inintraabdominal fat mass with severe obesity appear to bedue to higher numbers of adipocytes [4].The data on obese human HSL activity are also controver-sial, mainly because of methodological differences among theavailable studies [5]. Some authors demonstrated that theexpression and function of HSL in subcutaneous adipocytesare defective in obese patients. This HSL defect isaccompanied by a decreased lipolytic capacity of adipocyteswhen lipolysis is expressed per gram of lipid in fat cells.Lipolysis per gram of lipid is a better measure of lipolyticcapacity than lipolysis per cell, as fat cell volume increasesin obesity. Moreover, there is a relationship between cell sizeand the lipolysis rate per cell but not per gram of lipid [6].Discrepant data have been reported regarding the effect of weight loss on these enzymes. Bariatric procedures (withor without restrictive components) universally result in animprovement of metabolic syndrome (dyslipidemia, hyper-tension, insulin resistance, and central obesity). In addition,the procedure also improves hypertension. In light of thismarked amelioration of the components of the metabolicsyndrome accompanied by reliable maintenance of weight loss, malabsorptive bariatric procedures are the treatment of choice for morbid obesity and its comorbidities [7]. Someauthors observed that when obese subjects lost weight and became less hyperinsulinemic, adipose LPL continued toincrease. This suggests abnormal LPL regulation indepen-dent of insulin [5]. Others reported that weight loss did not change the lipolytic capacity of adipocytes or their capacityfor storing TAG [8, 9]. These incongruities may be related to (1) the way the enzyme activities were expressed (tissueweight, protein, DNA, etc.) and (2) the difficulties inmeasuring LPL activity since part of the enzyme remains buried in the tissue extracts. In contrast, measures of HSLactivity give the total amount of the enzyme in tissue [8].The aim of this study was to determine the LPL and HSLactivities in SAT and VAT depots from morbidly obese patients and compare them with a normal weight group(control). In addition, we wanted to study SAT from obese patients after bariatric surgery and compare it with that of the control group. Methods Subjects and Sample AcquisitionA group of 34 morbidly obese patients (24 women and tenmen) between 27 and 61 years of age with a BMI>40 kg/m 2 were recruited from the Vall d'Hebron Hospital in Barcelona,Spain, in accordance with the Spanish consensus on thediagnosis of obesity [10]. Twenty-five percent of womenand 20% of men had type 2 diabetes mellitus. The study protocol was accepted by the hospital's ethics committee,and all subjects gave their written, informed consent to participate.Two days before the study, all subjects were placed on anisocaloric diet calculated on the basis of individual require-ments. The diet was made up of 50% carbohydrate, 20% protein, and 30% fat. Blood samples were taken under fastingconditions between 8:00 and 10:00 A.M.  and at the time of surgery. Plasma was separated immediately by centrifuga- OBES SURG (2009) 19:1150  –  1158 1151  tion, and aliquots were frozen at   − 80°C for subsequent analysis. In obese subjects, subcutaneous abdominal adiposetissue (SAT) and visceral (epiploon) adipose tissue (VAT) biopsies were performed during Roux-en-Y gastric bypass(in tables and graph, obese). Second and third SAT biopsieswere obtained six and 16.3±3 months (range, 12  –  18 months)after bariatric surgery (in tables and graph, 6M or 12M,respectively). The anesthetic procedure was standardized for electivesurgeryand for the biopsyprocedures (1% scandicainwas used). Epinephrine was avoided. Tissue samples werequickly minced then frozen in liquid nitrogen and stored at  − 80°C for further analysis. Nine euthyroid patients (12 h fasted) who underwent alaparotomy (cholecystectomy or surgery of abdominalhernia) were used as controls. A blood sample and SATand VAT biopsies were taken simultaneously (in tables andgraph, control).Anthropometric and Body Composition MeasurementsBody weight, excess weight, height, and waist and hipcircumferences were measured according to standardized procedures [11]. Body mass index (BMI) and the waist-to-hip ratio (WHR) were then calculated. The body fat  percentage was calculated from the equation proposed byDeurenberg et al. [12], and the amount of total, subcutane-ous, and visceral fat were calculated from the equations proposed by Bonora et al. [13].Sample Preparation for LPL and HSL AssaysSAT and VAT homogenates for LPL and HSL enzymaticassays were obtained in a Polytron homogenizer at 4°Cwith 20  –  30 mg tissue/mL of freshly made buffer at pH7.4(10 mM HEPES, 1 mM EDTA, 1 mM DTT) with (for theLPL assay) or without (for the HSL assay) 5 U/mL heparin.Homogenates were centrifuged in a microfuge at 16,100×  g  for 10 min at 4°C, and the fat-depleted infranatants wereused for the enzymatic assays [14, 15]. LPL Activity AssayMethods involving the use of TAG containing radiolabeledacyl chains are highly specific and sensitive lipase assays[14, 16]. In our experimental procedure, LPL was assayed as previously described by Ballart et al. [17]. Theendothelial lipase described has no TAG hydrolase activitywhen 3  –  5% serum is present in the assay [18].HSL Activity AssayHSL was assayed as previously described by Ramírez et al.and Stam and Hülsmann [19] and [20], respectively]. The recently described enzyme adipose triglyceride lipase(ATGL) showed diminished TAG hydrolase activity in vitro,and its expression was unaffected by obesity and weight reduction [21].Lipid Extraction and DeterminationFor the lipid extraction, we applied our recently describedmethod to a small amount of tissue [22]. Cholesterol (freeand esterified), phospholipids, nonesterified fatty acids(NEFA), and TAG were measured immediately by enzy-matic analyses.Other DeterminationsProtein was determined in both plasma and adipose tissuehomogenates using the method developed by Bradford [23].DNA was quantified in liver biopsies by the fluorimetricmethod described by Vytasek [24]. Fasting plasma glucoselevels were measured enzymatically at the hospital's chem-istry laboratory using routine methods. Fasting plasmainsulin levels were determined by a commercial radioimmu-noassay kit (Amersham, Little Chalfont, UK). The homeo-stasis model assessment of insulin resistance (HOMA-IR)was calculated as previously described [25].Statistical AnalysisResults were reported as mean±SEM. Statistical signifi-cance of mean value differences for obese or normalweight, 6 or 12 months after surgery (weight loss), wasassessed by one-way ANOVA. Individual comparisonswere made using the Tukey's multiple comparison test.Correlations between independent variables were deter-mined by Spearman's correlation coefficient. Statisticalcomparisons were significant when  p <0.05.All statistical analyses were computed using the GraphPadPrism program, version 4.00 for Windows (GraphPadSoftware, San Diego, CA, USA, www.graphpad.com). Results Clinical Characteristics of PatientsThe characteristics of patients are shown in Table 1. Oneyear after bariatric surgery, the total percentage of weight loss was 37%, excess weight 71%, waist circumference27%, hip circumference 19%, and WHR 5.6%.Of the total body weight, 61% corresponded to total fat.Of this, 74% corresponded to subcutaneous fat and 27% tovisceral fat. The percentage of fat loss during the first 6 months after bariatric surgery was, approximately, the 1152 OBES SURG (2009) 19:1150  –  1158  same for total, subcutaneous, and visceral fat (48%, 43%,and 52%, respectively, all of them with  p <0.001 vs obese).Six months later, the percentage of fat loss was 59%, 55%,and 62% for total, subcutaneous, and visceral fat, respec-tively (  p <0.001 vs obese).In morbidly obese cases, body weight and excess weight correlated positively (  p <0.001) with total fat, visceral fat,BMI, as well as hip and waist measurements. Subcutaneousfat correlated positively with body weight (  p <0.05) andexcess weight (  p <0.001).These correlations persisted throughout the weight loss period (  p <0.001). Moreover, WHR correlated positivelywith body weight (  p <0.001) and excess weight (  p <0.05).Plasma Biochemical ParametersAs can be seen in Table 2, all parameters except cholesterolin low-density lipoprotein (cLDL) were significantly higher in the plasma of the morbidly obese subjects than in thecontrols. However, 6 months after surgery, the controlvalues had recovered, except in the case of NEFA, whichremained high even 1 year after surgery.Glycemia and insulin diminished by 29% and 63%,respectively, during the first 6 months. These levels weremaintained for the rest of the study period. The referencevalues for HOMA-IR which indicate insulin resistance,were around 3.5  –  3.8 [25]. Patients with morbid obesity had Table 2  Plasma parameters of control or obese patients before bariatric surgery and 6 and 12 months after surgeryControl Obese Time after bariatric surgery  p  (ANOVA)6M 12MTC (mg/dL) 164±3 205±9 a, * 169±9  b, ** 162±5  b, ** 0.0002cHDL (mg/dL) 74±4 47±2 a, * 45±2 a, * 51±2 a, * <0.0001cLDL (mg/dL) 105±4 128±6 103±7  b, *** 92±4  b, * 0.0014 NEFA (mg/dL) 9±0 16±3 a, *** 15±2 16±3*** <0.0001TAG (mg/dL) 89±9 147±12 a, *** 115±9  b, *** 95±6  b, * 0.0009Glyc (mg/dL) 1.0±0.0 0.4 ±0.1 0.3±0.0 0.2±0.0 0.0247Gluc (mg/dL) 72±2 126±10 a, * 89±4  b, * 91±3  b, * <0.0001Insulin (UI/L) 11±1 24±3 a, ** 9±1  b, * 8±1  b, * <0.0001HOMA-IR 2.0±0.1 8±1 a, ** 2±0  b, * 2±0  b, * <0.0001Data are expressed as mean±SEM. Statistical significance was calculated by one-way ANOVA and the Tukey ’ s post-test between groups TC   total cholesterol, cHDL cholesterol in HDL,  cLDL  cholesterol in LDL,  NEFA  nonesterified fatty acid,  TAG   triacylglycerides,  Glyc  glycerol, Gluc  glucose,  HOMA-IR  homeostasis model assessment-insulin resistant *  p <0.001; **  p <0.01; ***  p <0.05 a  Differences from control  b Differences in obese subjects vs 6 (6M) or 12 (12M) months after surgeryObese Time after bariatric surgery  p  (ANOVA)6M 12M N 34Sex 24 women and 10 menAge (years) 45±2Height (cm) 160±2Body weight (kg) 130±3 93±3 a, * 82±3 a,b, * , ** <0.0001BMI (kg/m 2 ) 49±1 35±1 a, * 31±1 a,b, * , *** <0.0001Excess weight (kg) 65±3 29±2 a, * 19±2 a,b, * , ** <0.0001Waist (cm) 136±2 105±5 a, * 99±3 a, * <0.0001Hip (cm) 145±2 124±5 a, * 118±4 a, * <0.0001Total fat (kg) 79±3 44 ±2 a, * 35±2 a,b, * , ** <0.0001Subcutaneous fat (kg) 58±3 35±3 a, * 31±2 a, * <0.0001Visceral fat (kg) 21±1 13±2 a, *** 11±2 a, *** 0.0002 Table 1  Clinical characteristicsof obese patients before bariatricsurgery and 6 and 12 monthsafter surgeryResults are expressed asmean±SEM. Statisticalsignificance was calculated byone-way ANOVA and theTukey ’ s post-test between groups  BMI   body mass index,  n  number of patients*  p <0.001; **  p <0.05; ***  p <0.01 a  Differences between obese and6 (6M) or 12 (12M) monthsafter surgery  b Differences between 6 and 12months after surgeryOBES SURG (2009) 19:1150  –  1158 1153  values four times higher than controls. Interestingly,6 months after bariatric surgery, HOMA-IR had diminished by 75% to control values and remained at this level for therest of the study period.In morbidly obese subjects, insulin correlated positivelywith body weight (  p <0.05). After bariatric surgery, insulinand HOMA-IR correlated positively with BMI, bodyweight, waist, excess weight, and total fat (  p <0.001) aswell as with WHR (  p <0.01), hip, and SAT (  p <0.05).Finally, VAT correlated with insulin (  p <0.05) and HOMA-IR (  p <0.01).Tissue Biochemical ParametersTable 3 shows a decrease in obese SAT and VAT DNAcontent per gram of tissue compared with controls (  p <0.01).Additionally, the amount of protein in SAT decreased 50%(  p <0.01) but remained stable in VAT. These data indicatethat obese SAT and VAT cells are bigger compared withcontrol ones. This is corroborated by the lipid content per DNA (or per cell), which is higher if it is assessed as totallipid (  p <0.01) in both SAT and VAT, or if we consider eachtype of lipid separately  —  except for PL. Taking this intoaccount, VATcells were smaller than SATcells in both obese(  p <0.001) and controls (  p <0.05)During weight loss, obese adipose tissue cells becamesmaller as their DNA content per gram of tissue increased progressively. Similarly, as the cell decreased in size, itslipid content also diminished. It is noteworthy that NEFAdecreased 38% 6 months after surgery, but from 6 to12 months, the decrease was only about 10%.LPL ActivityLPL activity in SAT and VAT expressed per gram of tissuewas similar (Fig. 1, upper left panel). However, obese SATvalues were up to four times higher than those observed incontrols (  p <0.001 vs control) despite having less cells per gram of tissue (see Table 3). Moreover, obese subjects alsohad more LPL activity per cell than controls (  p <0.001,Fig. 1, middle left panel). In VAT, although the cells weresmaller, obese subjects had 5.4 times more LPL activitythan controls (  p <0.001). If BW was taken into account (Fig. 1, lower left panel, note that activity is expressed in μ  mol OA body weight/min), SAT from obese patients hadan increased TAG storage capacity 7.6 times higher thancontrols (  p <0.001). In VAT, TAG storage capacity was 4.2times higher than controls (  p <0.001). Taken together, thiscould trigger the observed TAG accumulation described inTable 3.After bariatric surgery, SAT LPL activity per gram of tissue decreased. At 6M, it was still slightly higher thancontrols (  p <0.05), but at 12M, it had practically normal-ized. With weight loss, cells were smaller and exhibited less Table 3  SAT and VAT parameters of control or obese patients before surgery and 6 and 12 months after surgeryTime after surgeryControl Obese 6M 12M  p  (ANOVA)DNA ( μ  g/g tissue) SAT 94.3±4.4 68.9±2.5 a, * 76.0±7.2 131.5±17.1  b,c, * <0.0001VAT 107.6±3.5 d, ** 84.5±3.9 a,d, * , ***  – –  PROT (mg/  μ  g DNA) SAT 0.22±0.05 0.12±0.01 a, *** 0.26±0.04  b, * 0.27±0.09  b, ** 0.0035VAT 0.14±0.02 0.12±0.01  – –  Total lipids (mg/  μ  g DNA) SAT 7.09±0.74 11.06±0.44 a, * 8.83±0.77  b, ** 5.55±1.22  b,c, * , ** <0.0001VAT 5.39±0.62 9.04±0.67 a,d, ** , ***  – –  TAG (mg/  μ  g DNA) SAT 4.00±0.49 7.17±0.28 a, * 6.17±0.65 3.72±1.08  b,c, * , ** <0.0001VAT 3.11±0.41 6.33±0.48 a, ***  – –   NEFA (mg/  μ  g DNA) SAT 0.50±0.08 1.00±0.09 a, *** 0.62±0.07  b, *** 0.56±0.08  b, * , *** <0.0001VAT 0.33±0.04 0.61±0.07 a,d, ** , ***  – –  PL (mg/  μ  g DNA) SAT 2.02±0.19 2.03±0.14 1.20±0.16  b, * 0.91±0.19  b, * <0.0001VAT 1.50±0.19 1.38±0.19 d, ***  – –  TC (mg/  μ  g DNA) SAT 0.56±0.06 0.86±0.06 a, ** 0.94±0.10 0.54±0.12  b,c, ** 0.0096VAT 0.45±0.06 0.72±0.08  – –  Data are expressed as mean±SEM. Statistical significance was calculated by one-way ANOVA and the Tukey ’ s post-test between groups  PROT   proteins,  TAG   triacylglycerides,  NEFA  nonesterified fatty acids,  PL  phospholipids,  TC   total cholesterol*  p <0.001; **  p <0.05; ***  p <0.01 a  Differences from control  b Differences in obese subjects vs 6 (6M) or 12 (12M) months after surgery c Differences between 6 and 12 months after surgery d Differences between SAT and VAT1154 OBES SURG (2009) 19:1150  –  1158
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